Eukaryotic cells contain various membranes and membrane-bounded organelles that perform specialized functions. Protein targeting and transport systems are the fundamental processes that maintain these compartments. The importance of faithful protein targeting to human health is evident from the existence of diseases, such as I-cell and hyperoxaluria type I, where enzymes are localized to the wrong cellular compartments. The long range goal of our research is to understand the molecular mechanisms involved in the various protein transport systems. The work proposed here will investigate a curious phenomenon, whereby three evolutionarily related, but distinct systems target sub-populations of proteins to the same membrane, the thylakoid membrane of chloroplasts. Experiments are proposed to examine the three phases of the transport process for these systems: targeting, initial membrane insertion, and chain translocation across the bilayer. Specific in vitro experiments will identify and dissect the specific elements of targeting sequences that commit a preprotein to pathway. An analysis of the proposed receptors of these signals for two pathways, a chloroplast SecA homologue and a chloroplast SRP homologue, will assess their binding specificity and the chloroplast location where binding occurs. Other experiments will attempt to determine the molecular makeup of the chloroplast SRP. Finally, studies of the events at the membrane will characterize initial insertion across the bilayer and polypeptide chain translocation. In particular, experiments are designed to determine if these three pathways have distinct membrane components of if they merge at the level of a common pore. These latter studies will employ in vivo experiments to reduce the amount of one suspected pore component, a chloroplast SecY homologue, by the expression of antisense RNA. Genetic studies of the yeast ER, bacterial cytoplasmic membrane, and plant thylakoid membrane have revealed the essential in vivo nature of multiple protein targeting pathways to the same membrane. The successful completion of the experiments proposed here will describe the molecular basis for this phenomenon and provide the foundation necessary for experimentally addressing the reason for multiplicity of protein targeting pathways?